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Expression of Peanut Chlorotic Streak Caulimovirus (PClSV) Gene VI in Escherichia Coli: Detection of Product in Infected Plants Using Gene VI Protein Specific Antibodies
Indu B. Maiti, Narayan C. Das and Aparajita Chatterjee

The gene VI product of the Caulimoviruses is a multifunctional protein. In an effort to better characterize the Peanut chlorotic streak caulimovirus (PClSV), a newly described member of Caulimovirus group, the PCR generated coding sequence of PClSV-geneVI (nucleotide position 4618 to 5880 in the PClSV genome) was cloned into an Escherichia coli expression vector pET15b under the control of T7 promoter. The E. coli strain BL21(DE3) pLysS harboring the resulting plasmid pET15bPVI, upon induction with 0.04 mM of IPTG, synthesized a protein of apparent MW of 49 kD, an expected size for the PClSV-gene VI protein, as detected by SDS-PAGE. The over produced protein was sequestered in inclusion bodies. The protein was purified to homogeneity by elctroeluting from gel. Polyclonal antibodies against purified PClSV-gene VI protein were raised in the rabbit. In Western blot analysis it was shown that rabbit antisera cross-reacted with PClSV-gene VI protein from PClSV infected plants or PClSV gene VI transfected protoplasts. However, it did not cross-react with gene VI protein of Cauliflower mosaic virus (CaMV) or Figwort mosaic virus (FMV).

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